The present invention relates to a Daucus carota reduced pigment gene, a carrot seed, a carrot plant, a carrot inbred and a method of producing carrot hybrids. The reduced pigment gene of the present invention can be incorporated into various Daucus genetic backgrounds. The present invention also relates to a carrot root having an increased level of xcex1-tocopherol.
Carrot (Daucus carota L.) is a biennial plant that belongs to the parsley family. Carrot roots are commonly-known as a good source of Vitamin A. In particular, it has been estimated that carrots contribute approximately 14% of the total Vitamin A to the human diet in the United States (Senti, F. R., and R. L. Rizek, 1975, Nutrient Levels in Horticultural Crops. Hort.Science. 10:243-246). Vitamin A content is related to the pigmentation in the carrot roots. In particular, carrot roots contain xcex2-carotene which animals convert into provitamin A. Beta carotene is also responsible for the orange color of carrot roots. Carrot pigmentation is present in carrots in many different forms. Carrot roots can exhibit several colors including white, yellow, orange, red and purple (Banga, 0., 1964, Origin and Distribution of the Western Cultivated Carrot. Genetica Agrafia. 17:357-370). Of these colors, purple pigmentation is due to the presence of anthocyanins whereas yellow, orange and red pigmentation are due to carotenoids. The primary carotenoids in orange carrot tissue are xcex1 and xcex2-carotene (Laferriere, L., and W. H. Gabelman, 1968, Inheritance of Color, Total Carotenoids, Alpha-carotene, and Beta-carotene in Carrots, Daucus carota L., Proc. Amer. Soc. Hort. Sci. 93:408-418).
Carrot cultivars are often separated into several categories for market use. These include 1) fresh market, 2) cut and peel, and 3) processing. Fresh market carrots are typically known as Imperator types and have long, straight, thin roots. They are also known as cello or bunching carrots because they are sold bunched in cello bags in the market. Cut and peel carrots refer to the xe2x80x9cbabyxe2x80x9d carrot now seen in markets throughout the world. These carrots have roots that are similar in type to the fresh market carrot, however, they have been cut into small sections for market. Processing carrots are large, often tapered, bulky roots used for canning, freezing, and other processed carrot products. Cultivars of processed carrot and fresh market carrot are developed and maintained in separate breeding programs.
Although the beta-carotene present in commonly-known carrots may be converted into Vitamin A in the body, sufficient levels of other nutrients must be obtained from sources other than carrots. For example, the presence of xcex1-tocopherol has not been recorded in any carrot inbred, hybrid, or openpollinated cultivar. Furthermore, because xcex1-tocopherol is usually associated with the oil fraction of plant extracts, (while carrots are consumed for their high moisture content and high fiber root) carrot inbred lines, hybrids, or open-pollinated cultivars have not been associated with this vitamin. Alpha tocopherol (Vitamin E) cannot be synthesized by the body. Alpha tocopherol is important in the body as a vitamin. Beyond its importance as a vitamin, xcex1-tocopherol also possesses antioxidant activity. More specifically, xcex1 tocopherol along with other members of the Vitamin E family, namely, xcex2, xcex3, and xcex4 tocopherol and their corresponding unsaturated derivatives xcex1, xcex2, xcex3 and xcex4 tocotrienol, are primarily used by the body as antioxidants. Nevertheless, the average U.S. citizen consumes less than the U.S. Recommended Daily Allowance of 30 International Units of Vitamin E. Biosynthetic pathway for carotenoids (left column) and tocopherols (right column) are shown in Table 1 below:
Biosynthesis of carotenoids and tocopherols is connected through the common intermediate geranylgeranyl pyrophosphate (GGPP), Norris et al., 1995, Plant Cell, 7:2139-2149.
Although some vegetables synthesize both xcex1-tocopherol and xcex2-carotene, these vegetables are primarily seed-producing plants such as maize and some seed oil plants. It would be desirable to have a new carrot that synthesizes xcex1 tocopherol (Vitamin E). Moreover, it would be desirable to have a carrot inbred line with xcex1 tocopherol synthesis in its root.
The present invention relates to a Daucus seed, a Daucus plant, a Daucus variety, a Daucus hybrid and a method for producing a Daucus plant.
More specifically, the invention relates to a carrot root having a mutant reduced pigment gene designed rp. The present invention is directed to a carrot root with a total xcex1-tocopherol content between about 0.01 mg per 100 grams of fresh weight of the carrot root and about 0.40 mg per 100 grams of fresh weight of the carrot root. The present invention is also directed to an F1 hybrid carrot plant having a total xcex1-tocopherol content greater than about 0.01 mg per 100 grams of fresh weight of the carrot root. The present invention further relates to a method of producing the disclosed carrot plants and seeds by crossing a reduced pigment plant of the instant invention with another carrot plant. The invention also relates to the transfer of the genetic reduced pigment into other genetic backgrounds.
It is an object of this invention to provide a carrot that synthesizes xcex1-tocopherol in its root. Generally, the carrot inbred line of the present invention provides for xcex1-tocopherol biosynthesis in the root. The carrot seed of the present invention contains a recessive gene, designated rp, for a reduced pigment phenotype. The present invention is also directed to a reduced pigment carrot plant produced from growing the carrot seed. The carrot plant has an increased level of xcex1-tocopherol. The xcex1-tocopherol level of the carrot plant is at least 0.01 mg per 100 grams of fresh weight of the carrot. In general, a method of the present invention is for producing F1 hybrid carrots. This method includes crossing a first parent carrot plant with a second parent carrot plant. The resultant F1 hybrid carrot root is harvested. Either the first or second parent carrot plant is the reduced pigment carrot plant produced by growing the seed which contains the gene (allelic DNA genetic factor) for reduced pigment phenotype of the present invention. A first generation (F1) hybrid carrot plant is produced by growing the hybrid carrot root produced by the method of the present invention.
In order to provide an understanding of some of the terms used in the specification and claims, the following definitions are provided:
xcex1-tocopherolxe2x80x94as used herein, the term alpha(xcex1)-tocopherol is synonymous with Vitamin E.
Daucus carotaxe2x80x94as used herein, the term Daucus carota is synonymous with carrot.
Although trace levels have been speculated, no detected levels of xcex1-tocopherol have been confirmed in Daucus carota until the present invention. Additionally, there are no known reports of xcex1-tocopherol biosynthesis in any Daucus carota species, cultivar, in the wild or commercially available. The novel xcex1-tocopherol biosynthesis of the present invention arose from breeding and research efforts which were conducted beginning in 1996.
The instant invention is the genetic expression of a mutant reduced pigment gene. This reduced pigment gene is associated with increased xcex1-tocopherol biosynthesis. The genetic basis for xcex1-tocopherol production in Daucus carota involves a single recessive allele. When the reduced pigment gene is incorporated into different genetic backgrounds of Daucus carota in the homozygous recessive condition, the xcex1-tocopherol characteristic is transferred into these genetic backgrounds.
The seeds from the developed true-breeding reduced pigment lines can also be marketed. Reduced pigment lines can also be used as one of the parents in F1 hybrid seed production resulting in an F1 hybrid.
The reduced pigment gene has been designated rp. The rp gene was the result of a spontaneous mutation in the inbred line W266D. The resulting mutant was designated W266E (also called W266Erprp). The total carotenoid content in this mutation is approximately 95% less than found in other processing carrots. As discussed more fully below, analysis of this mutation led to the determination that the mutant synthesizes xcex1 tocopherol (Vitamin E) at a low rate.
Several white carrot roots were discovered during the propagation of the carrot inbred line W266D. These roots were identical in shape and size to their orange counterparts (W266D), however their roots lacked pigment. The non-pigmented roots remained pale during early growth stages and developed a slight yellow color in the phloem and outer xylem at maturity. This mutant line was called xe2x80x98E-Whitexe2x80x99. The reduced pigment trait was genetically analyzed. This reduced pigment phenotype was believed to be conditioned by a single recessive gene. The symbol rp was used to describe the genetic control of this xe2x80x98reduced-pigmentxe2x80x99 phenotype. Mature roots of the rprp genotype were harvested at 120 days after planting. The rprp roots exhibited a whitish-yellowish appearance and contained 141 xcexcg carotene per gram of dry weight. In comparison, the orange-pigmented roots of W266D at the same growth stage contained almost 1800 xcexcg carotene per gram of dry weight. Thus, it was determined that the rp gene does not completely block carotenoid synthesis since mature roots from rprp plants exhibited small amounts of xcex2 carotene.
In addition to providing a reduced pigment phenotype, the transfer of the recessive gene to different genetic backgrounds has produced the associated characteristics of increased xcex1-tocopherol when present as a double recessive rprp. Analysis of chromatographs of extracts of the rprp roots showed a unique peak at approximately twenty-two minutes. This peak was not present in extracts of orange-rooted carrots (such as W266D) and was unlike any known carotenoid in its absorption maximum. The extracts of the rprp roots were further examined and compared to pure xcex1-tocopherol using a high performance liquid chromatograph (HPLC) with a diode array detector. The comparison highlighted the similarity of the root peak and the xcex1-tocopherol peak. Further chromatographic analysis was performed and large volumes of rprp root extract was prepared and injected into a liquid chromatograph. The peak at twenty-two minutes was collected in several fractions, dried, and analyzed, in comparison with a pure standard of xcex1-tocopherol by GC-Mass Spectrometry. The data confirmed that the unique peak in the rprp extract was xcex1-tocopherol. The production of xcex1-tocopherol is not limited to the non-orange carrots. In the F1 hybrid, the rp gene is in the heterozygous condition (RPrp). This specific hybrid has an orange carrot root and has a total carotenoid content that is generally not significantly different from the wild type (RPRP).
The mutant gene of the present invention can be easily transferred to other carrot inbreds and other genetic backgrounds by making an initial cross and selecting for the gene using standard breeding procedures. Parental lines have been developed which when crossed produce an F1 hybrid which has a double recessive for the 1p gene. The xcex1-tocopherol level in the plant and roots has ranged upwards to approximately 0.40 mg per 100 grams of fresh weight.
The present invention is directed to a carrot root with a total xcex1-tocopherol content between about 0.01 mg per 100 grams of fresh weight of the carrot root and about 0.40 mg per 100 grams of fresh weight of the carrot root. The present invention is also directed to an F1 hybrid carrot plant having a total xcex1-tocopherol content greater than about 0.01 mg per 100 grams of fresh weight of the carrot root.
As used herein, the terms xe2x80x9cplantxe2x80x9d includes plant cells, plant protoplasts, plant cells of tissue culture from which carrot plants can be regenerated, plant calli, plant clumps, and plant cells that are intact in plants or parts of plants such as pollen, flowers, seeds, leaves, stems and the like. Tissue culture of carrot is described in Simon, P. W. 1985. Use and improvement of carrot for genetic studies, p. 194-198. In: M. Terzi, L. Pitto and R. Sung (eds.). Somatic embryogenesis of carrots. Consiglio Nazionale delle Richerche, Incremento Produttivita Risorse Agricole, Rome; and Simon, P. W. 1984. Carrot genetics. Plant Molecular Biology Reporter, 2:54-63, incorporated herein by reference.